The present invention relates to a novel recombinant plasmid inserted with a Hepatitis B virus gene, an yeast transformed with the plasmid, and a method for the production of Hepatitis B virus surface antigen. More particularly, it relates to a recombinant plasmid which is obtained by inserting Hepatitis B virus (HBV) surface antigen (hereinafter, referred to as "HBs antigen", "HBsAg" or "s antigen") gene into a shuttle vector which can replicate in both Escherichia coli and yeast at downstream of the expression control region of the repressible acid phosphatase gene (said region being hereinafter referred to as "acid phosphatase promoter" or "acid phosphatase gene") carried on the vector, an yeast transformed with said recombinant plasmid, and a method for the production of an immunologically active HBs antigen (HBsAg) comprising culturing said transformed yeast in an appropriate medium under the conditions that the acid phosphatase promoter is not repressed.
Hepatitis B which is usually caused by transfusing blood of HBV positive patient or others is hardly remedied, and there is no drug suitable for complete remedy thereof. Most suitable prophylaxis is a vaccine consisting of HBs antigen. However, it is very difficult to produce the HBsAg vaccine in an industrial scale, because HBV is infectious only to human subjects and chimpanzee (it has never been succeeded to make cell culture infected with HBV), and owing to this specificity of HBV, HBsAg must be obtained only from human blood serum.
It has recently been proposed to prepare HBsAg by E. coli with a recombinant DNA instead of using human blood serum (cf. Japanese Patent Laid Open Application No. 104887/1980). However, according to this method using E. coli, it is still difficult to produce the desired HBsAg in an industrial scale, because the produced HBsAg is easily decomposed within the cells of E. coli and further growth of E. coli is inhibited by the HBsAg produced, which results in less productivity of HBsAg.
It has very recently been reported that HBs antigen particles have successively been produced by an yeast [cf. Nature, 298, 347-350 (22 July, 1982)]. It is described in this report to recombine a gene encoding an HBs protein to an E. coli-yeast shuttle vector at downstream of an alcohol dehydrogenase I (ADH1) promoter which is usually used in the production of interferone by yeast. According to this method, however, the amount of HBs protein is so small, and hence, it is not satisfactory to produce the desired HBs antigen in an industrial scale.
The present inventors have extensively studied on an improved method for producing HBsAg in an industrial scale. As a result, it has been found that the desired HBsAg can be produced in a large scale by recombining a gene of HBs antigen to a specific plasmid vector having an yeast gene and E. coli gene and carrying the repressible acid phosphatase gene of the yeast including the phosphatase promoter, transforming an yeast with the recombinant DNA thus obtained, and culturing the transformed yeast, and that the HBsAg thus obtained has the same immunological properties as those of HBsAg originated from human blood plasma.
An object of the present invention is to provide a novel recombinant plasmid inserted with an HBs antigen gene. Another object of the invention is to provide a transformed yeast which is produced by transforming an yeast with the novel recombinant plasmid as set forth above. A further object of the invention is to provide a method for production of HBsAg in an industrial scale. These and other objects and advantages of the invention will be apparent to persons skilled in the art from the following description.
The recombinant plasmid inserted with an HBV gene of the present invention is obtained by utilizing a shuttle vector which has both E. coli gene and yeast gene and can replicate in both of them, and by recombining an HBs antigen gene into the vector to downstream of the acid phosphatase promoter carried on the vector wherein a part or whole of the structural gene of the acid phosphatase or a certain region of upstream of the phosphatase structural gene are preferably deleted. The transformed yeast is prepared by transforming an yeast with the thus obtained plasmid which expresses HBs gene by a conventional method. The desired HBs protein can be produced by culturing the transformed yeast in an appropriate medium, preferably under the conditions that the acidic phosphatase promoter is not repressed.